Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome.

Department of Anatomy and Neurobiology, University of California, Irvine, California 92697, USA.

Abstract

Fragile X syndrome, the most commonly known genetic cause of autism, is due to loss of the fragile X mental retardation protein, which regulates signal transduction at metabotropic glutamate receptor-5 in the brain. Fragile X mental retardation protein deletion in mice enhances metabotropic glutamate receptor-5-dependent long-term depression in the hippocampus and cerebellum. Here we show that a distinct type of metabotropic glutamate receptor-5-dependent long-term depression at excitatory synapses of the ventral striatum and prefrontal cortex, which is mediated by the endocannabinoid 2-arachidonoyl-sn-glycerol, is absent in fragile X mental retardation protein-null mice. In these mutants, the macromolecular complex that links metabotropic glutamate receptor-5 to the 2-arachidonoyl-sn-glycerol-producing enzyme, diacylglycerol lipase-α (endocannabinoid signalosome), is disrupted and metabotropic glutamate receptor-5-dependent 2-arachidonoyl-sn-glycerol formation is compromised. These changes are accompanied by impaired endocannabinoid-dependent long-term depression. Pharmacological enhancement of 2-arachidonoyl-sn-glycerol signalling normalizes this synaptic defect and corrects behavioural abnormalities in fragile X mental retardation protein-deficient mice. The results identify the endocannabinoid signalosome as a molecular substrate for fragile X syndrome, which might be targeted by therapy.

(a) Alignment of the coding region in human (h), mouse (m) and rat (r). DGL-α mRNAs reveal a putative G-quartet sequence within a G-rich region containing several DWGG repeats. The canonical G-quartet motif is DWGG-N(0–5)-DWGG-N(0–3)-DWGG-N(0–2)-DWGG, where D is any nucleotide except C, and W is A or U. The DWGG repeats are boxed in red. (b) Co-immunoprecipitation of FMRP with DGL-α mRNA. Brains from wild-type or fmr1−/− mice were homogenized and centrifuged at 70,000 g for 30 min. The supernatant (1 mg protein) was incubated with the indicated amounts of anti-FMRP antibody or normal serum (NS), and the immunocomplex was precipitated using protein G-sepharose beads. Top, levels of DGL-α mRNA in the immunoprecipitates were quantified by real-time quantitative PCR (n=3, ***P<0.001). Bottom, a portion of the immunoprecipitate was subjected to SDS–PAGE and western blot analyses to confirm the presence of FMRP. (c,d) Analyses of mRNAs encoding for DGL-α, DGL-β and NAPE-PLD (c) or positive control PSD-95 and amyloid precursor protein (d) in anti-FMRP-immunoprecipitates (n=3, **P<0.01 and ***P<0.001 compared with NS, ##P<0.01 and ###P<0.001 compared with DGL-α-anti-FMRP). Similar results were obtained using fmr1−/− mice bred on either a FVB.129 background (b,c) or a C57BL/6J background (d). (e) DHPG-induced dissociation of DGL-α mRNA from FMRP in cultured cortical neurons. Rat primary neurons were prepared from embryonic day 18 cortex, as described. Cells were treated with DHPG (100 μM) in culture medium and harvested at the indicated time. Levels of FMRP-bound DGL-α mRNA were determined by anti-FMRP immunoprecipitation and quantitative PCR, as described in Methods (n=5, ***P<0.001). Results are representative of at least two independent experiments. Significance was determined using two-tailed Student's t-test. Error bars represent s.e.m.

(a) Synaptoneurosome (SN) fractions prepared from wild-type or fmr1−/− mice (1 mg ml−1) were incubated at 37 °C for 30 min in the presence of 100 μM DHPG, and DGL activity was measured in vitro using 10 μM diheptadecanoylglycerol as a substrate (n=4). (b) DGL activity was measured in SN fractions after incubation with various concentrations of DHPG (n=3). (c) 2-AG levels in SN fractions were measured after 45-min incubation with DHPG (100 μM) or vehicle (Veh, PBS) (n=5–9). (d). After treatment of synaptoneurosomes with DHPG, as described above, levels of DGL-α protein were measured by western blot. Representative images for DGL-α and loading control actin are shown (n=6 each). Experiments were conducted on fmr1−/− mice on C57BL/6J (a,d) or FVB.129 background (b,c). Results are representative of at least two independent experiments. Significance was determined using two-tailed Student's t-test. *P<0.05, **P<0.01 and ***P<0.001. Error bars represent s.e.m.

(a) Summary graph showing whole-cell evoked EPSC amplitudes. Values are normalized to baseline before the induction of LTD and averaged per minute. Tetanic stimulation (10 min at 10 Hz starting at time 0) of PFC afferent fibres to medium spiny neurons in the ventral striatum induces a robust LTD in wild-type littermates (open symbols, n=4) but not fmr1−/− mice (filled symbols, n=5). Here as in all physiology figures, n equals the number of animals. (b) Summary graph showing averaged time courses of the experiments in which the 10 min at 10-Hz protocol was given in control ACSF (open symbols, n=4) and after pre-treatment with tetrahydrolipstatin (THL, 10 μM, filled symbols, n=4), an inhibitor of the DGL-α. Graphs show EPSC amplitudes normalized to baseline before the induction of LTD and averaged per minute. (c) Direct pharmacological activation of mGlu1/5 with 50 μM (S)-DHPG induces LTD in the ventral striatum of wild-type littermates (open symbols, n=16) but not fmr1−/− mice (filled symbols, n=21). Summary graphs show excitatory postsynaptic field potentials (fEPSP) amplitudes. (d) Summary graph showing fEPSP amplitudes recorded in pyramidal neurons of the PFC. Values are normalized to baseline before the induction of LTD and averaged per minute. Tetanic stimulation (10 min at 10 Hz starting at time 0) of layers 2/3 to layers 5/6 pyramidal synapses induces a robust LTD in wild-type littermates (open symbols, n=8) but not fmr1−/− mice (filled symbols, n=5). Statistical significance was determined using Mann–Whitney U-test. Error bars represent s.e.m.

Effects of the MGL inhibitor JZL184 on open field (a,b) and elevated plus maze (c,d) behaviours. Wild-type and fmr1−/− mice received JZL184 (JZL, 16 mg kg−1, intraperitoneal) or vehicle (Veh), and were tested 6 h after injections. Activity in the open field was measured by counting the number of squares crossed (a) and total distance travelled (b). In the elevated plus maze test, we measured percentage of open arm entries (c) and average time in open per each entry (d). Significance was determined using two-way analysis of variance with post-hoc Student–Newman–Keuls test. NS, not significant; *P<0.05, and ***P<0.001 compared with wild-type-Veh; #P<0.05, and ##P<0.01 compared with fmr1−/−-Veh (n=11 per each group). Error bars represent s.e.m.